The soluble form of the membrane-bound transferrin homologue, melanotransferrin, inefficiently donates iron to cells via nonspecific internalization and degradation of the protein.

Melanotransferrin (MTf) is a membrane-bound transferrin (Tf) homologue found particularly in melanoma cells. Apart from membrane-bound MTf, a soluble form of the molecule (sMTf) has been identified in vitro[Food, M.R., Rothenberger, S., Gabathuler, R., Haidl, I.D., Reid, G. & Jefferies, W.A. (1994) J. Biol. Chem.269, 3034-3040] and in vivo in Alzheimer's disease. However, nothing is known about the function of sMTf or its role in Fe uptake. In this study, sMTf labelled with 59Fe and 125I was used to examine its ability to donate 59Fe to SK-Mel-28 melanoma cells and other cell types. sMTf donated 59Fe to cells at 14% of the rate of Tf. Analysis of sMTf binding showed that unlike Tf, sMTf did not bind to a saturable Tf-binding site. Studies with Chinese hamster ovary cells with and without specific Tf receptors showed that unlike Tf, sMTf did not donate its 59Fe via these pathways. This was confirmed by experiments using lysosomotropic agents that markedly reduced 59Fe uptake from Tf, but had far less effect on 59Fe uptake from sMTf. In addition, an excess of 56Fe-labelled Tf or sMTf had no effect on 125I-labelled sMTf uptake, suggesting a nonspecific interaction of sMTf with cells. Protein-free 125I determinations demonstrated that in contrast with Tf, sMTf was markedly degraded. We suggest that unlike the binding of Tf to specific receptors, sMTf was donating Fe to cells via an inefficient mechanism involving nonspecific internalization and subsequent degradation.     

Iron uptake from transferrin and transferrin endocytic cycle in Friend erythroleukemia cells

Several aspects of iron metabolism were studied in cultured Friend erythroleukemia cells before and after induction of hemoglobin synthesis by dimethyl sulfoxide. The maximal rate of iron uptake from 59Fe-labeled transferrin, 1.5 X 10(6) atoms of Fe/cell per 30 min in uninduced cells, increased to 3 X 10(6) atoms/cell after 5 days of induction. The increase in iron uptake was not accompanied by a proportional increase in the number of transferrin receptors detected by 125I-labeled transferrin binding, suggesting a more efficient iron uptake by transferrin receptors in induced cells, with the rate of about 26 iron atoms per receptor per hour, compared to 15 atoms in uninduced cells. In agreement with this conclusion are results of the study of cellular 125I or 59Fe labeled transferrin kinetics. In the induced cells transferrin endocytosis and release proceeded with identical rates and all the endocytosed iron was retained inside the cell. On the other hand, transferrin release by uninduced cells was significantly slower and a substantial part of internalized 59Fe was released. On the basis of these results, different efficiency of iron release from internalized transferrin, accompanied by changes in cellular transferrin kinetics, is proposed as one of the factors determining the rate of iron uptake by developing erythroid cells.     

Iron and gallium increase iron uptake from transferrin by human melanoma cells: further examination of the ferric ammonium citrate-activated iron uptake process

Previously we showed that preincubation of cells with ferric ammonium citrate (FAC) resulted in a marked increase in Fe uptake from both (59)Fe-transferrin (Tf) and (59)Fe-citrate (D.R. Richardson, E. Baker, J. Biol. Chem. 267 (1992) 13972-13979; D.R. Richardson, P. Ponka, Biochim. Biophys. Acta 1269 (1995) 105-114). This Fe uptake process was independent of the transferrin receptor and appeared to be activated by free radicals generated via the iron-catalysed Haber-Weiss reaction. To further understand this process, the present investigation was performed. In these experiments, cells were preincubated for 3 h at 37 degrees C with FAC or metal ion solutions and then labelled for 3 h at 37 degrees C with (59)Fe-Tf. Exposure of cells to FAC resulted in Fe uptake from (59)Fe-citrate that became saturated at an Fe concentration of 2.5 microM, while FAC-activated Fe uptake from Tf was not saturable up to 25 microM. In addition, the extent of FAC-activated Fe uptake from citrate was far greater than that from Tf. These results suggest a mechanism where FAC-activated Fe uptake from citrate may result from direct interaction with the transporter, while Fe uptake from Tf appears indirect and less efficient. Preincubation of cells with FAC at 4 degrees C instead of 37 degrees C prevented its effect at stimulating (59)Fe uptake from (59)Fe-Tf, suggesting that an active process was involved. Previous studies by others have shown that FAC can increase ferrireductase activity that may enhance (59)Fe uptake from (59)Fe-Tf. However, there was no difference in the ability of FAC-treated cells compared to controls to reduce ferricyanide to ferrocyanide, suggesting no change in oxidoreductase activity. To examine if activation of this Fe uptake mechanism could occur by incubation with a range of metal ions, cells were preincubated with either FAC, ferric chloride, ferrous sulphate, ferrous ammonium sulphate, gallium nitrate, copper chloride, zinc chloride, or cobalt chloride. Stimulation of (59)Fe uptake from Tf was shown (in order of potency) with ferric chloride, ferrous sulphate, ferrous ammonium sulphate, and gallium nitrate. The other metal ions examined decreased (59)Fe uptake from Tf. The fact that redox-active Cu(II) ion did not stimulate Fe uptake while redox-inactive Ga(III) did, suggests a mechanism of transporter activation not solely dependent on free radical generation. Indeed, the activation of Fe uptake appears dependent on the presence of the Fe atom itself or a metal ion with atomic similarities to Fe (e.g. Ga).     

Iron uptake by Chinese hamster fibroblasts from human transferrin

The manner of uptake or iron by Chinese hamster fibroblasts, type DON, from human transferrin was investigated by means of replacement studies, in which the cells that were incubated with ¹²⁵I-labelled human transferrin were chased with non-radioactive transferrin for only a few minutes. The results did not support the reversible endocytosis hypothesis for the uptake of iron from transferrin. The uptake of iron measured as ⁵⁹Fe during several cell divisions was found to be a function of time and cell number. It was found that the total uptake of iron in the harvests was directly proportional to the incubation, and that the uptake per 10⁶ cells levelled off in the course of time.     

Inhibition of tumor growth by a truncated and soluble form of melanotransferrin

Melanotransferrin is a glycoprotein expressed at the cell membrane and secreted in the extracellular environment. Recombinant truncated form of membrane-bound melanotransferrin (sMTf) was reported to exert in vitro anti-angiogenic properties. Here we show that sMTf treatment leads to a 50% inhibition of neovascularization in Matrigel implants when stimulated by growth factors. Using a glioblastoma xenograft model, we demonstrate that sMTf delivery at 2.5 and 10 mg/kg/day by micro-osmotic pump inhibits tumor growth by 73% and 91%, respectively. In a lung carcinoma xenograft model, sMTf treatment at 2.5 and 10 mg/kg/day impeded tumor growth by 87% and 97%. Furthermore, subcutaneous glioblastoma and lung carcinoma tumors from mice treated with 10 mg/kg/day of sMTf present insignificant growth toward the study. In association with a reduction in endoglin mRNA expression, the hemoglobin content decreased by half in sMTf-treated glioblastoma tumors. In vitro experiments revealed that NCI-H460 cells treated with sMTf display an inhibition in their invasive capabilities with a concomitant reduction in the expression of the low-density lipoprotein receptor protein and urokinase plasminogen activator receptor. Altogether, our results demonstrate that sMTf exerts anti-cancer and anti-angiogenic activities, suggesting that its administration may provide novel therapeutic strategies for the treatment of cancer.     

Iron uptake and transfer from ceruloplasmin to transferrin

Background

Dietary and recycled iron are in the Fe^2 + oxidation state. However, the metal is transported in serum by transferrin as Fe^3 +. The multi-copper ferroxidase ceruloplasmin is suspected to be the missing link between acquired Fe^2 + and transported Fe^3 +.

Methods

This study uses the techniques of chemical relaxation and spectrophotometric detection.

Results

Under anaerobic conditions, ceruloplasmin captures and oxidizes two Fe^2 +. The first uptake occurs in domain 6 (< 1 ms) at the divalent iron-binding site. It is accompanied by Fe^2 + oxidation by Cu^2 +_D6. Fe^3 + is then transferred from the binding site to the holding site. Cu⁺_D6 is then re-oxidized by a Cu^2 + of the trinuclear cluster in about 200 ms. The second Fe^2 + uptake and oxidation involve domain 4 and are under the kinetic control of a 200 s change in the protein conformation. With transferrin and in the formed ceruloplasmin–transferrin adduct, two Fe^3 + are transferred from their holding sites to two C-lobes of two transferrins. The first transfer (~ 100 s) is followed by conformation changes (500 s) leading to the release of monoferric transferrin. The second transfer occurs in two steps in the 1000–10,000 second range.

Conclusion

Fe^3 + is transferred after Fe^2 + uptake and oxidation by ceruloplasmin to the C-lobe of transferrin in a protein–protein adduct. This adduct is in a permanent state of equilibrium with all the metal-free or bounded ceruloplasmin and transferrin species present in the medium.

General significance

Ceruloplasmin is a go-between dietary or recycled Fe^2 + and transferrin transported Fe^3 +.     

Iron uptake by Chang cells from transferrin,nitriloacetate and citrate complexes: The effects of iron-loading and chelation with desferrioxamine

Iron uptake by Chang liver cells in culture is about thirty times as great when ferric nitriloacetate is used as a donor as when iron-transferrin is used. Iron uptake from ferric citrate is no greater than from iron-transferrin. Most of the intracellular iron derived from transferrin is found in the supernatant after 20 000 × g centrifugation of the cell homogenate for 40 min: about half of this is in the form of ferritin. Iron derived from ferric nitriloacetate is found largley in the membranous pellet after centrifugation and very little of this is in the form of ferritin.Iron incorporated in cytosol ferritin is easily available for chelation by desferrioxamine and this process is facilitated by ascorbic acid. Membrane-bound iron is less available for chelation. This tissue culture model forms a convenient basis for the study of iron overlead and iron chelation.     

Iron and transferrin uptake by phytohemagglutinin-stimulated human peripheral blood lymphocytes

Iron (Fe) and transferrin (TF) uptake by human peripheral blood lymphocytes stimulated in vitro with phytohemagglutinin was measured. Pulses of 59FeTF or 125I-TF were added to the cultures either at time 0 or 8 hr before the end of a 72-hr incubation. In time-course experiments, peak iron and transferrin uptake coincided with the peak of tritiated thymidine uptake taken as a measure of cellular activation. Iron, but not transferrin, was accumulated by the cells. Non-linear relationships existed between both iron and transferrin uptake and the degree of activation. Both rose markedly above basal levels only at a level of activation at least 50% of the maximum observed. The results suggest that although iron utilization is related to cellular activity, the uptake mechanism is only activated when an increased iron metabolism has exhausted internal stores.     

Two mechanisms of iron uptake from transferrin by melanoma cells. The effect of desferrioxamine and ferric ammonium citrate.

The effects of ferric ammonium citrate (FAC) and desferrioxamine (DFO) on iron (Fe), and transferrin (Tf) uptake have been investigated using SK-MEL-28 human melanoma cells, which express the Tf homologue, melanotransferrin, in high concentrations. Previously we demonstrated two separate Fe uptake mechanisms from Tf, viz. a specific process mediated by the transferrin receptor (TfR) and a nonspecific process (Richardson, D. R., and Baker, E. (1990) Biochim. Biophys. Acta 1053, 1-12). Cells exposed to DFO demonstrated up-regulation of the TfR with a concurrent increase in the rate of Fe uptake. Desferrioxamine also stimulated the nonspecific process of Fe uptake, resulting in a further increase in accumulation of Fe over Tf after saturation of the specific TfR. Ferric ammonium citrate had two effects. First, it resulted in down-regulation of the TfR. Second, and paradoxically, it markedly stimulated the rate of Fe uptake from Tf by the nonspecific process without increasing the rate of nonspecific Tf uptake. These data conclusively demonstrate that two entirely different mechanisms of iron uptake from Tf exist in melanoma cells and that ferric ammonium citrate may be a useful experimental tool to further characterize the specific and nonspecific mechanisms of Fe uptake from Tf.     

Iron uptake by glial cells

Dynamic studies of iron metabolism in brain are generally unavailable despite the fact that a number of neurologic conditions are associated with excessive accumulation of iron in central nervous tissue. Cortical non-neuronal (glial) cultures were prepared from fetal mouse brain. After 13 days the cultures were exposed to radiolabeled iron. Brisk and linear total iron uptake and ferritin iron uptake occurred over 4 hours. When methylamine or ammonium chloride was added, (both known inhibitors of transferrin iron release because of their lysosomotropic properties), total iron uptake was diminished. Further studies indicated that meth-ylamine inhibits glial cell ferritin iron incorporation. Glial cell iron transport is similar to previously reported neuronal cell iron transport (1) but glial cell iron uptake proceeds at a faster rate and is more susceptible to the inhibition of certain lysosomotropic agents. The data reinforces the likelihood that iron uptake by nervous tissues is transferrin-mediated.     

The transferrin homologue, melanotransferrin (p97), is rapidly catabolized by the liver of the rat and does not effectively donate iron to the brain

Melanotransferrin (MTf) or melanoma tumor antigen p97 is a membrane-bound transferrin (Tf) homologue that binds iron (Fe). This protein is also found as a soluble form in the plasma (sMTf) and was suggested to be an Alzheimer's disease marker. In addition, sMTf has been recently suggested to cross the blood-brain barrier (BBB) and accumulate in the brain of the mouse following intravenous infusion. Considering the importance of this observation to the physiology and pathophysiology of the BBB and the function of sMTf in vivo, we investigated the uptake and distribution of 59Fe-125I-sMTf and compared it to 59Fe-125I-Tf that were injected intravenously in rats. Studies were also performed to measure 59Fe and 125I-protein uptake by reticulocytes using these radiolabelled proteins. The results showed that sMTf was rapidly catabolized, mainly in the liver and to a lesser extent by the kidneys. The 59Fe was largely retained by these organs but the 125I was released into the plasma. Only a small amount of 125I-sMTf or its bound 59Fe was taken up by the brain, less than that from 59Fe-125I-Tf. There was much less 59Fe uptake by erythropoietic organs (spleen and femurs) from 59Fe-sMTf than from 59Fe-Tf, and no evidence of receptor-mediated uptake of sMTf was obtained using reticulocytes. It is concluded that compared to Tf, sMTf plays little or no role in Fe supply to the brain and erythropoietic tissue. However, a small amount of sMTf was taken up from the plasma by the brain and a far greater amount by the liver.     

The role of the membrane-bound tumour antigen, melanotransferrin (p97), in iron uptake by the human malignant melanoma cell.

Melanotransferrin (MTf) is a membrane-bound transferrin (Tf) homologue with several characteristics in common with serum Tf. MTf is found at high levels in melanoma cells and previous studies have shown that MTf can bind Fe. In addition, Chinese hamster ovary cells transfected with MTf transport Fe from 59Fe-citrate at greater rates than control cells. However, the role of MTf in the Fe uptake process of human melanoma cells remains unknown. In the present study we have characterized the role of MTf in Fe uptake by SK-Mel-28 melanoma cells in order to understand its function. Initial studies examined whether modulation of intracellular Fe levels using the Fe chelator desferrioxamine (DFO) or the Fe donor ferric ammonium citrate (FAC) could change MTf mRNA levels. In contrast to transferrin receptor (TfR) mRNA that increased after exposure to DFO and decreased after incubation with FAC, there was no change in MTf mRNA levels. In addition, compared to control cells, there was no alteration of 125I-labelled anti-MTf mAb-binding in cells exposed to DFO or FAC, suggesting no change in the number of MTf sites. Further studies examined the ability of DFO and FAC to modulate Fe uptake from 59Fe-citrate which is bound by MTf. In contrast to the effect of DFO or FAC at increasing and decreasing Fe uptake from 59Fe-Tf, respectively, DFO had no influence on 59Fe-citrate uptake, whereas FAC markedly increased it. Collectively, these studies suggest that MTf is not regulated in a manner similar to the TfR in response to cellular Fe levels. MTf can be removed from the membrane by phosphatidylinositol-specific phospholipase C (PtdIns-PLC). Preincubation of melanoma cells with PtdIns-PLC reduced anti-MTf mAb binding to 3% of the control, while PtdIns-PLC only slightly reduced 59Fe uptake from 59Fe-citrate. These results suggest that MTf played only a minor role in Fe uptake from 59Fe-citrate by these cells. The expression of MTf mRNA (poly A+) was also examined in 50 human tissues and found to be markedly different to Tf mRNA or TfR mRNA. Surprisingly, MTf mRNA expression was widespread in normal tissues, and was observed at its highest levels in the salivary gland. In contrast to expectations, MTf mRNA expression was generally greater in adult than fetal tissues.     

Iron mobilization from ultraviolet-irradiated,iron-saturated,transferrin

SUMMARY

Transferrin is the major iron transport protein of mammalian plasma. The ultraviolet-B irradiation of 1.4 mg/ml iron saturated transferrin solutions (~32 μM Fe³⁺) induces a Fe³⁺ loss accompanied by Fe²⁺ formation. The initial quantum yield of Fe³⁺ loss is wavelength dependent (φ(313 nm)~1.3×10^?3) and oxygen independent suggesting an intramolecular electron transfer from one of the Fe³⁺ ligands. A photolysis of tryptophan residues parallels this photoreduction.     

脑内的铁,转铁蛋白及转铁蛋白受体

脑铁异常增高可能参与脑神经变性疾病的发生发展。这一发现使得脑铁代谢成为近年广为关注和研究较为广泛的领域。本文综述了这一领域某些方面的目前认识。包括：（１）脑铁分布及功能;（２）铁转铁蛋白及转铁蛋白受体在脑内的合成与分布;（３）脑铁摄取和运输。此外,对铁与某些金属离子,转的蛋白和转铁蛋白受体与脑神经变性疾病的关系,以及转铁蛋白受体内吞在生物大分子跨血脑屏障运输中的作用也作了简要讨论。     

Inhibition of melanoma brain metastasis by targeting melanotransferrin at the cell surface

Brain metastases are a common feature of malignant melanoma and are associated with poor prognosis. Melanotransferrin (MTf), one of several antigens associated with the surface of melanoma cells, has been demonstrated to promote cell invasion. In this study, we investigated the role of membrane‐bound MTf in several of the steps leading to the development of melanoma brain metastasis. Our results indicated that MTf‐positive cells were detected in the brains of nude mice injected intravenously with human melanoma SK‐Mel 28 cells. Moreover, administration of a single dose of a monoclonal antibody (L235) directed against human MTf significantly reduced the development of human melanoma brain metastases in nude mice. The ability of melanoma cells to cross the blood–brain barrier (BBB) in vitro is correlated with their MTf expression levels at the cell surface. Overall, our results indicated that membrane‐bound MTf is a key element in melanoma cell transmigration across the BBB and subsequent brain metastasis. Thus, these data suggest MTf as an attractive target and demonstrate the therapeutic potential of an anti‐MTf mAb for preventing metastatic melanoma.     

Effect of glycosylation on the function of a soluble, recombinant form of the transferrin receptor

Production of the soluble portion of the transferrin receptor (sTFR) by baby hamster kidney (BHK) cells is described, and the effect of glycosylation on the biological function of sTFR is evaluated for the first time. The sTFR (residues 121-760) has three N-linked glycosylation sites (Asn251, Asn317, and Asn727). Although fully glycosylated sTFR is secreted into the tissue culture medium ( approximately 40 mg/L), no nonglycosylated sTFR could be produced, suggesting that carbohydrate is critical to the folding, stability, and/or secretion of the receptor. Mutants in which glycosylation at positions 251 and 727 (N251D and N727D) is eliminated are well expressed, whereas production of the N317D mutant is poor. Analysis by electrospray ionization mass spectrometry confirms dimerization of the sTFR and the absence of the carbohydrate at the single site in each mutant. The effect of glycosylation on binding to diferric human transferrin (Fe(2) hTF), an authentic monoferric hTF with iron in the C-lobe (designated Fe(C) hTF), and a mutant (designated Mut-Fe(C) hTF that features a 30-fold slower iron release rate) was determined by surface plasmon resonance; a small ( approximately 20%) but consistent difference is noted for the binding of Fe(C) hTF and the Mut-Fe(C) hTF to the sTFR N317D mutant. The rate of iron release from Fe(C) hTF and Mut-Fe(C) hTF in complex with the sTFR and the sTFR mutants at pH 5.6 reveals that only the N317D mutant has a significant effect. The carbohydrate at position 317 lies close to a region of the TFR previously shown to interact with hTF.     

Iron transfer from transferrin to ferritin mediated by pyrophosphate

There is no significant iron exchange from transferrin to ferritin in the absence of reducing and chelating agents. Pyrophosphate can release iron from transferrin and can be isolated as a ferric pyrophosphate complex by ion exchange chromatography. We have established that pyrophosphate alone can mediate iron exchange from transferrin to ferritin. Under these conditions, iron is incorporated directly into ferritin as Fe(III).     

Escape mechanisms of melanoma from immune system by soluble melanoma antigen

We demonstrate in the B16 melanoma (C57BL/6 derived) system that the soluble form of tumor Ag preferentially suppresses immune responses 1) by inhibiting CTL activity in the effector phase and 2) by induction of specific Ts that block CTL generation in the induction phase. Soluble melanoma antigen Ag injected i.p. into the tumor-bearing host can effectively augment melanoma growth in vivo. Two T cell types with the L3T4+ or double-negative/I-J+ phenotype are involved in the suppression of anti-melanoma CTL responses and can easily be generated in the in vitro primary 12 h-culture. Anti-melanoma Ts recognizes the GM3(NeuAc) structure and distinguishes GM3 molecular species. This is because liposomes constructed with GM3(NeuAc) but not with GM3(NeuGc) gangliosides alone can effectively induce the melanoma-specific Ts. It is thus likely that tumor cells can escape from the immunologic surveillance system by stimulating the repertoire of Ts for self-Ag, GM3, which has existed even in the unprimed conditions in order to maintain self-tolerance. These would appear to be the major escape mechanisms.